Enhanced CTL epitope-containing HIV-1 reverse transcriptase polypeptides

ABSTRACT

The present invention provides peptides and proteins for use in second generation HIV vaccines and as diagnostic tools in the treatment and control of HIV infection. The antiviral protection shown by compositions of the present invention has not been previously achieved with an HLA epitope-enhanced vaccine. These findings define a critical balance between MHC affinity and receptor crossreactivity required for effective epitope enhancement and also demonstrate construction and efficacy of such a component of a new generation vaccine.

This application is a U.S. National Phase of PCT/US2004/009617, filedMar. 29, 2004, which claims the benefit of U.S. provisional patentapplication No. 60/459,507, filed on Mar. 31, 2003, the disclosures ofwhich are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to the fields of immunology and genetics,particularly with regard to HIV infection and prevention of the same.The present invention provides peptides and proteins for use in secondgeneration HIV vaccines and as diagnostic tools in the treatment andcontrol of HIV infection. The antiviral protection shown by compositionsof the present invention has not been previously achieved with an HLAepitope-enhanced vaccine. These findings define a critical balancebetween MHC affinity and receptor crossreactivity required for effectiveepitope enhancement and also demonstrate construction and efficacy ofsuch a component of a new generation vaccine.

BACKGROUND OF THE INVENTION

Protection by classical vaccines such as polio vaccine is mediatedmostly by neutralizing antibodies, but such antibody-inducing vaccineshave been ineffective against viruses causing chronic infection such asHIV or hepatitis C virus. Rather, in this case, T-cell immunity might becrucial as has been confirmed by CD8 cell depletion in AIDS virusinfection of macaques (Schmitz, J. B., M. et al., (1999), Science283:857; and Jin, X., et al., (1999), J. Exp. Med. 189:991her, viralsequences evolving under immune selective pressure would not likely haveoptimal HLA molecule-binding epitopes. Thus, modifying epitope sequenceto improve the CTL response could be one effective strategy towarddevelopment of new generations of vaccines (Berzofsky, J. A., et al.,(1999) Immunol. Rev. 170:151; and Berzofsky, J. A., et al., (2001)Nature Reviews Immunology 1:209).

CD8 cytotoxic T cells (CTL) play a major role in protection against HIVor SIV virus (Musey, L., J. et al., (1997) N Engl J Med 337:1267;Schmitz, J. E., et al., (1999), Science 283:857; and Jin, X., et al.,(1999), J. Exp. Med. 189:991.). Nevertheless, the natural immuneresponse to HIV is often unable to clear the infection. Although anumber of antigens that induce CTL responses and can help to eliminateor reduce virus production by killing viral producer cells have beenreported so far, these do not seem to be sufficient to eliminateinfection in most cases. There is no reason to expect that the HIVsequence would have evolved to have optimal CTL epitopes to alloweradication of the virus.

We have previously succeeded in improving the affinity of a hepatitis Ccore epitope for HLA-A2.1 (Sarobe, P., et al., (1998), J. Clin. Invest.102:1239) and of a helper epitope for murine class II MHC (Ahlers, J.D., et al., (1997), Proc. Natl. Acad. Sci. U.S.A. 94:10856; and Ahlers,J. D., et al., (2001), J. Clin. Invest. 108:1677), and anepitope-enhanced melanoma peptide has shown efficacy in human clinicaltrials (Rosenberg, S. A., et al., (1998) Nature Medicine 4:321). Othercomplementary approaches to improve affinity for T-cell receptors havebeen devised (Zaremba, S., et al., (1997) Cancer Res 57:4570; andSlansky, J. E., et al., (2000) Immunity 13:529; and Tangri, S., et al.,(2001) J Exp Med 194:833). Although one substitution resulting in higheraffinity HLA binding of another HIV peptide has been reported (Pogue, R.R., J. et al., (1995) Proc. Natl. Acad. Sci. U.S.A. 92:8166), nosystematic attempt to improve epitopes of HIV has been carried out. Inparticular, no systematic analysis of the competing effects ofsubstitutions on HIV peptide binding to the HLA class I molecule vspeptide/HLA complex binding to the T cell receptor has been reported.

Further, to our knowledge, protection against viral infection in vivo byan epitope-enhanced vaccine mediated by CTL restricted by a human HLAmolecule has not previously been demonstrated.

SUMMARY OF THE INVENTION

In principle it should be possible to improve the immunogenicity ofepitopes, a process called “epitope enhancement,” to develop a morehighly effective HIV vaccine (Berzofsky, J. A., et al., (1999), Immunol.Rev. 170:151; and Berzofsky, J. A., et al., (2001), Nature ReviewsImmunology 1:209). Using epitope enhancement, we have developednon-natural peptides and proteins having utility as the active agents insecond generation vaccines and as diagnostic tools in the treatment andprevention of HIV-1 infection. The present invention also includesmethods for using these vaccines and reagents.

Accordingly, the present invention provides immunostimulating peptideshaving an amino acid sequence X₁LYQYMDDV (SEQ ID NO:1), where X₁ is anyhydrophobic amino acid. This amino acid sequence motif, which to ourknowledge is not found in nature, is common to all peptides and proteinsof the invention, and preferably has the amino acid sequence VLYQYMDDV(SEQ ID NO:2) or YLYQYMDDV (SEQ ID NO:3).

As noted above, immunostimulating proteins are also provided by thepresent invention, for example, one embodiment provides animmunostimulating peptide or protein comprising the sequenceX₁X₂LYQYMDDVX₃ (SEQ ID NO:4) where X₁ is a sequence of amino acidresidues of between 0 and 200 residues in length; X₂ is any hydrophobicamino acid; and, X₃ is a sequence of amino acid residues of between 0and 200 residues in length. Preferably, these immunostimulating proteinshave the sequence X₁VLYQYMDDVX₃ (SEQ ID NO:5), or X₂YLYQYMDDVX₃ (SEQ IDNO:6).

A further embodiment provides proteins and fusion molecules having theamino acid sequence motif X₁LYQYMDDV (SEQ ID NO:1), where X₁ is anyhydrophobic amino acid. The fusion molecules may include, for example,HIV-1 viral proteins, glycolipid conjugates, or conjugation of a proteinor peptide having the X₁LYQYMDDV (SEQ ID NO:1) sequence motif with animmunostimulating carrier protein, as described herein. In someembodiments, the fusion molecules include repeat (“concatameric”)X₁LYQYMDDV (SEQ ID NO:1) sequences. Concatamers of the present inventionare particularly efficient both as protein/peptide antigens and, whenprovided as encoding nucleic acid sequences, gene therapy reagents, asdescribed herein.

All peptides, proteins and fusion molecules of the present invention maybe modified as described herein. Modifications may be made for a varietyof reasons, for example, to increase solubility, cell uptake, or easeadministration as a medicament. Modifications contemplated as beingencompassed by the invention include N-terminal acetylation of peptides,C-terminal amidation, esterfication and reduction of the C-terminalamino acid carboxyl group; and glycosylation, amidation, acylation,esterfication, oxidation or rediction of aminoacyl side chain residues,as is known in the art. (see, e.g., Techniques in Protein Modificationand Analysis pp. 151-154, 1995).

The present invention also provides immunostimulating peptides andproteins in medicament form. These embodiments contain, at a minimum, atherapeutically effective amount of one or more of the immunostimulatingpeptides and proteins discussed above, with a pharmaceuticallyacceptable excipient. Exemplary excipients are discussed in detail,below. The medicaments may optionally include immunostimulant(s) tofurther enhance their therapeutic value.

Other medicaments provided by the invention include an immunostimulatingpeptide or protein, or a nucleic acid encoding the same, pulsed ortransduced into dendritic cells.

Nucleic acid vaccines, including “live” vaccines are also contemplatedby the present invention. For example, the invention providesmedicaments that contain vector(s) having a nucleic acid that includes anucleotide sequence encoding one of the immunostimulatory peptides orproteins discussed above. Some of the vetors of the present inventionmay include coding nucleotide sequences for more than one of theimmunostimulating peptides and proteins, including transcriptional unitsfor producing more than one of the immunostimulating peptides andproteins by, for example placing an IRES sequence before downstreamcoding sequences. Methods for forming constructs of this type are wellknown to those of skill in the art.

Introducing a nucleic acid vaccine of the present invention to a subjectresults in expression of the nucleic acid, thereby inducing an immuneresponse in the subject directed against an epitope of a product(s)encoded by the nucleic acid.

Nucleic acid medicaments of the present invention include naked nucleicacids, virus and bacterial vectors. Construction and delivery methodsfor these types of vaccines are known. (see, e.g., U.S. Pat. Nos.6,534,483; 6,495,318; 6,475,995; Drobnitz, J., Advanced Drug DeliveryReviews, vol. 3, 229-245, 1989; Kuo, P. Y. P. et al., Critical Reviewsin Eukaryotic Gene Expression, vol. 6, No. 1, pp. 59-73, 1996; Hopkinset al. Infect Immun. 63:3279-3286, 1995; Srinavasin et al. Vaccines 95,R N. Chanock et al., Eds., Cold Spring Harbor Laboratory Press,Plainview, N.Y., p 273-280, 1995).

Another embodiment of the invention are methods for preventing ortreating an HIV-1 infection that entail administering a dose of themedicament discussed above in an amount effective to induce an immuneresponse capable of preventing HIV-1 infection or reducing HIV-1 viralload in a patient. These medicaments are particularly suited for use inprimates, including humans.

An additional embodiment is methods of assessing immune function ordiagnosing exposure to HIV-1 for a subject. Performing these methodsinvolves, at a minimum, contacting a blood sample, including T cells,obtained from the subject cells with a peptide or protein of the presentinvention, then determining an immune response of the subject's T cellsto the peptide or protein. In some aspects, the determining step isperformed by assaying for RANTES or IFN-g production, or lysis of cellsdisplaying the peptide by cytotoxic T lymphocytes, or any combination ofthe three parameters, induced with the peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a comparison of the HLA-A2 binding curves among the wild typeRT (179-187), VIYQYMDDL (SEQ ID NO:7), RT-1Y (YIYQYMDDL; SEQ ID NO:8),RT-2L9V (VLYQYMDDV; SEQ ID NO:2), and RT-1Y2L9V (YLYQYMDDV; SEQ ID NO:3)in the T2-binding assay.

FIG. 1B is a comparison of the HLA-A2 binding curves among the RT-2L9V,p17-WT (SLYNTVATL; SEQ ID NO:9), RT-1Y2L9V and FMP (GILGFVFTL; SEQ IDNO:10).

FIG. 2A illustrates the recognition of RT-WT and RT-2L9V peptides byRT-WT and RT-2L9V specific CTL lines from A2Kb-transgenic mice as afunction of peptide concentration, revealing the difference in peptideaffinity for HLA-A2 and CTL avidity for the same peptide-MHC complexes.(E/T ratio, 10:1)

FIG. 2B illustrates the recognition of RT-WT, RT-1Y, RT-2L9V andRT-1Y2L9V peptides by RT-WT, RT-2L9V and RT-1Y2L9V specific CTL linesfrom HHD-2-transgenic mice as a function of peptide concentration,revealing the difference in peptide affinity for HLA-A2 and CTL avidityfor the same peptide-MHC complexes. (E/T ratio, 10:1)

FIG. 3A illustrates IFN-γ production by RT-WT and -2L9V specific CTLline derived from A2Kb-transgenic mice.

FIG. 3B illustrates IFN-γ production by RT-2L9V and -1Y2L9V specific CTLline derived from HHD-2-transgenic mice.

FIG. 4A illustrates RANTES production by RT-WT and RT-2L9V specific CTLline derived from A2Kb-transgenic mice.

FIG. 4B illustrates RANTES production by RT-2L9V and -1Y2L9V specificCTL line derived from HHD-2-transgenic mice.

FIG. 5A illustrates induction of CTL immune response and comparison ofCTL avidity against RT-WT in A2Kb-transgenic mice using different RTpeptide variants.

FIG. 5B illustrates induction of antigen specific IFN-γ production bypeptides-specific culture lines.

FIG. 6 graphically depicts the protection induced by immunization withRT-peptides.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

The terms “peptide” and “protein” are used herein to refer to a polymerof amino acid residues. The terms also apply to amino acid polymers inwhich one or more amino acid residue is an artificial chemical mimeticof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymer. Peptides and proteins of the present inventioninclude amino acid polymers having D- and L-isoforms of individual aminoacid residues, as well as other amino acid variants, as describedherein. Peptides are distinguished by the number of amino acid residuesmaking up the primary structure of the molecule. For purposes of thisinvention, peptides are those molecules comprising up to 50 amino acidresidues, and proteins comprise 50 or more amino acid residues. However,methods of synthesis and/or delivery of peptides and proteins of theinvention are similar, if not identical, as will be appreciated by oneof skill in the art. Therefore, where appropriate, these terms aresynonymous when discussing methods of synthesis, modification or use astherapeutic or diagnostic reagents.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and o-phosphoserine. “Amino acid analog” refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid. Amino acid mimetics refers to chemical compoundsthat have a structure that is different from the general chemicalstructure of an amino acid, but that function in a manner similar to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Amino acid sequence” refers to the positional relationship of aminoacid residues as they exist in a given polypeptide or protein.

“Hydrophobic amino acid” refers to amino acids, both natural andsynthetic, having a hydrophobicity value of 0.5 or greater.Hydrophobicity values are “scaled” values from computational log(P)determinations by the “Small Fragment Approach” (see, “Development ofHydrophobicity Parameters to Analyze Proteins Which Bear Post- orCotranslational Modifications” Black, S. D. and Mould, D. R. (1991)Anal. Biochem. 193, 72-82). The equation used to scale raw log(P) valuesto the scaled values given is as follows: Scaled Parameters=(RawParameters+2.061)/4.484.

Hydrophobicity values for naturally occurring amino acids are given intable 1, below.

TABLE 1 Designations for Unmodified L-alpha-Amino Acids Amino Acid3-Letter Code 1-Letter Code Hydrophobicity Alanine Ala A 0.616 CysteineCys C 0.680 Aspartate Asp D 0.028 Glutamate Glu E 0.043 PhenylalaninePhe F 1.00 Glycine Gly G 0.501 Histidine His H 0.165 Isoluecine Ile I0.943 Lysine Lys K 0.283 Leucine Leu L 0.943 Methionine Met M 0.738Asparagine Asn N 0.236 Proline Pro P 0.711 Glutamine Gln Q 0.251Arginine Arg R 0.000 Serine Ser S 0.359 Threonine The T 0.450 Valine ValV 0.825 Tryptophan Trp W 0.878 Tyrosine Tyr Y 0.880

“Aliphatic amino acid” refers to amino acids, both natural andsynthetic, having a hydrophobicity value of 0.5 or greater, and onlysaturated carbon-carbon bonds. Aliphatic amino acids include alanine,glycine, isoleucine, leucine, proline and valine.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-o-methyl ribonucleotides andpeptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions, see below) and complementary sequences,as well as the sequence explicitly indicated.

“Conservatively modified variants” refers to those nucleic acids thatencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical sequences. The term also refers to fragments of particularsequences, where the sequence of the fragment has been conservativelymodified as described herein. Because of the degeneracy of the geneticcode, a large number of functionally identical nucleic acids encode anygiven protein. For instance, the codons GCA, GCC, GCG and GCU all encodethe amino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein that encodes a polypeptide also describes every possiblesilent variation of the nucleic acid. One of skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine, and UGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid that encodes apolypeptide is implicit in each described sequence. (See e.g., Batzer etal., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.,260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98(1994)).

The term “coding sequence”, in relation to nucleic acid sequences,refers to a plurality of contiguous sets of three nucleotides, termedcodons, each codon corresponding to an amino acid as translated bybiochemical factors according to the universal genetic code, the entiresequence coding for an expressed protein, or an antisense strand thatinhibits expression of a protein. A “genetic coding sequence” is acoding sequence where the contiguous codons are intermittentlyinterrupted by non-coding intervening sequences, or “introns.” DuringmRNA processing intron sequences are removed, restoring the contiguouscodon sequence encoding the protein or anti-sense strand.

“Excipient” refers to an inert substance used as a diluent or vehiclefor a drug

“Immunostimulant”, and grammatical variants thereof, refer to anysubstance capable of stimulating an immune response.

An “immune response” is any physiological change resulting in activationand/or expansion of a “B” cell population with production of antibodies,and/or activation and/or expansion of a “T” cell population.

“T cell” refers to any lymphocyte that matures in the thymus and has theability to recognize specific peptide antigens, or specific peptideantigens complexed with a major histocompatability complex protein(MHC), through the receptors on its cell surface.

“Vector” refers to any type of genetic construct containing a nucleicacid capable of being transcribed in a cell. Vectors used for theamplification of nucleotide sequences (both coding and non-coding) arealso encompassed by the definition. In addition to the coding sequence,vectors will generally include restriction enzyme cleavage sites and theother initial, terminal and intermediate DNA sequences that are usuallyemployed in vectors to facilitate their construction and use. Theexpression vector can be part of a plasmid, virus, or nucleic acidfragment.

“Fusion molecules” refers to any molecule formed through the structurallinkage of a peptide of the present invention to one or more molecules,particularly macromolecules. In the context of the present inventionother molecules that can be joined to peptides of the invention to formfusion molecules include sugars and polysaccarides, other peptides andproteins, lipids, and nucleotides and nucleic acids.

“HIV-1 infection” refers to indications of the presence of the HIV-1virus in an individual including asymptomatic seropositivity,aids-related complex (arc), and acquired immunodeficiency syndrome(AIDS).

“HIV-1 viral load” refers to the number of viral particles in a sampleof blood plasma HIV viral load is increasingly employed as a surrogatemarker for disease progression. It is measured by PCR and bDNA tests andis expressed in number of HIV copies or equivalents per milliliter.

“IFN-γ” or “interferon-γ” refers to a cytokine elaborated by Tlymphocytes in response to either specific antigen or mitogenicstimulation.

DETAILED DESCRIPTION I. Introduction

The present invention provides immunostimulatory peptides and proteins,and the nucleic acids encoding them, for use as therapeutic anddiagnostic tools for the treatment of HIV infection. The peptides andproteins of the invention all share the same amino acid sequence motif,which is a variant of a synthetic sequence motif derived from the HIV-1reverse transcriptase catalytic site region. This motif has the sequenceX₁LYQYMDDV (SEQ ID NO:1), where X₁ is any hydrophobic amino acid.

The invention also provides second generation HIV vaccines and methodsfor their use. These vaccines have the immunostimulatory peptides andproteins of the invention, or the nucleic acids encoding them, as theiractive ingredients.

The peptides and nucleic acids of the present invention may also be usedas diagnostic reagents for determining the presence or monitoring theprogression of an HIV infection. For example, the nucleic acids of thepresent invention may be labeled with a detectable label, such as afluorescent or radioactive moiety and used as a hybridization probe todetect the presence of HIV nucleic acid in a body fluid of an infectedindividual. Failure to detect hybridization partners for such probeswould be indicative of absence of infection. Peptides and proteins ofthe invention may for example be used to test for an immune responseagainst the peptide or protein, as described herein.

As shown by the examples to this application, peptides of the presentinvention produce high avidity CTL. As high avidity CTL have been foundto be critical in clearance of virus infection (Alexander-Miller, M. A.,et al., (1996), Proc. Natl. Acad. Sci. U.S.A. 93:410; and Gallimore, A.,T. et al., (1998), J Exp Med. 187:1647) the ability of anepitope-enhanced peptide to induce high avidity CTL, as we have seenhere and with a hepatitis C virus peptide (Sarobe, P., C. D. et al.,(1998), J. Clin. Invest. 102:1239.), makes the molecules of the presentinvention attractive as vaccines. These molecules are applicable to allforms of vaccine, e.g., peptide, DNA, recombinant viral or bacterialvector, or live attenuated virus. They also define and demonstrate theefficacy of a prototype conserved enhanced epitope that can beincorporated into many candidate vaccines currently under study.

II. Producing Immunostimulatory Peptides

The present invention provides immunostimulating peptides with the aminoacid sequence X₁LYQYMDDV (SEQ ID NO:11), where X₁ is any hydrophobicamino acid, preferably valine. These immunostimulatory peptides may besynthesized by any of the techniques that are known to those skilled inthe peptide art, including recombinant DNA techniques and isolatednatural sources, such as whole viruses or tumors, which express proteinsthat include a segment having the amino acid sequence of the presentinvention.

Synthetic chemistry techniques, such as a solid-phase Merrifield-typesynthesis, are preferred for reasons of purity, antigenic specificity,freedom from undesired side products, ease of production and the like.Excellent summaries of the many techniques available can be found in J.M. Steward & J. D. Young, SOLID PHASE PEPTIDE SYNTHESIS, W.H. FreemanCo., San Francisco, (1969); M. Bodanszky et al., PEPTIDE SYNTHESIS, JohnWiley & Sons, Second Edition, (1976); and J. Meienhofer, HORMONALPROTEINS AND PEPTIDES, Vol. 2, p. 46, Academic Press, New York (1983)for solid phase peptide synthesis, and E. Schroder & K. Kubke, 1 THEPEPTIDES, Academic Press, New York (1965) for classical solutionsynthesis, each being hereby incorporated herein by reference.Appropriate protective groups usable in such synthesis are described inthe above texts and in J. F. W. McOmie, PROTECTIVE GROUPS IN ORGANICCHEMISTRY, Plenum Press, New York (1973), the entire disclosure of whichis also incorporated herein by reference. Simplified methods for solidphase synthesis of peptides on a small scale also are known. See forinstance, Houghten, R. A., Proc. Natl. Acad. Sci. U.S.A. 82:5131-5135(1985); and Houghton, M., Q.-L. Choo, & G. Kuo, European PatentApplication 88310922 (1988).

Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes an immunogenic peptide of interest isinserted into an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression. These procedures are generally known in the art, asdescribed generally in Sambrook et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982), whichis incorporated herein by reference.

Coding sequences for the immunostimulatory peptides and proteins of thepresent invention may be synthesized by chemical techniques, forexample, the phosphotriester method of Matteucci et al., J. Am. Chem.Soc. 103:3185 (1981), modification can be made simply by substitutingthe appropriate base(s) for those encoding the native peptide sequence.The coding sequence can then be provided with appropriate linkers andligated into expression vectors commonly available in the art, and thevectors used to transform suitable hosts to produce theimmunostimulatory peptide or protein. A number of such vectors andsuitable host systems are now available. For expression, the codingsequence will be provided with operably linked start and stop codons,promoter and terminator regions and usually a replication system toprovide an expression vector for expression in the desired cellularhost. For example, promoter sequences compatible with bacterial hostsare provided in plasmids containing convenient restriction sites forinsertion of the desired coding sequence. The resulting expressionvectors are transformed into suitable bacterial hosts. Of course, yeastor mammalian cell hosts may also be used, employing suitable vectors andcontrol sequences.

III. Producing Fusion Molecules Having an Immunostimulatory Amino AcidSequence

Although the peptides of the invention will preferably be substantiallyfree of contaminants, including naturally occurring host cell proteinsand fragments thereof, in some embodiments the peptides can besynthetically conjugated to native fragments or particles,immunostimulatory molecules and the like to form advantageous fusionmolecules. Both peptides and fusion molecules of the invention may be intheir neutral (uncharged) forms or in forms which are salts, and eitherfree of modifications such as glycosylation, side chain oxidation, orphosphorylation or containing these modifications, subject to thecondition that the modification not destroy the biological activity ofthe polypeptides as herein described.

One fusion molecule embodiment is a peptide or protein that includes inits amino acid sequence the sequence motif X₁X₂YQYMDDVX₃ (SEQ ID NO:23)where X₁ is a sequence of amino acid residues of between 0 and 200residues in length; X₂ is any hydrophobic amino acid; and, X₃ is asecond sequence of amino acid residues of between 0 and 200 residues inlength that may be different from the X₁ sequence.

Another fusion molecule embodiment of the invention can be aglycoprotein, lipoprotein, nucleoprotein or other heterologous moleculehaving the sequence motif X₁LYQYMDDV (SEQ ID NO:1), where X₁ is anyhydrophobic amino acid. Preferably this fusion protein will include anamino acid sequence for an HIV-1 viral protein or an immunostimulatingcarrier protein.

These fusion molecules may be produced by methods known to those ofskill in the art and are typically designed to improve antigenicity ofthe immunostimulatory peptide sequence included in the molecule, or aidin its delivery to a patient. Thus the peptides and fusion molecules ofthe present invention may be modified as necessary to provide certaindesired attributes, e.g., improved pharmacological characteristics,while increasing or at least retaining substantially all of thebiological activity of the unmodified peptide to bind the desired MHCmolecule and induce a CTL response.

For instance, the fusion molecules may be subject to various changes,such as substitutions, either conservative or nonconservative, wheresuch changes might provide for certain advantages in their use, such asimproved MHC binding. By conservative substitutions is meant replacingan amino acid residue with another that is biologically and/orchemically similar, e.g., one hydrophobic residue for another, or onepolar residue for another. The substitutions include combinations suchas Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr, Lys, Arg;and Phe, Tyr. The effect of single amino acid substitutions may also beprobed using D-amino acids. Such modifications may be made using wellknown peptide synthesis procedures, as described in e.g., Merrifield,Science 232:341-347 (1986), Barany and Merrifield, The Peptides, Grossand Meienhofer, eds. (New York, Academic Press), pp. 1-284 (1979); andStewart and Young, Solid Phase Peptide Synthesis, (Rockford, Ill.,Pierce), 2d Ed. (1984), incorporated by reference herein.

The peptides of the invention may also be modified by extending theiramino acid sequence, e.g., by the addition of amino acids to their N orC terminus. The peptides or fusion molecules of the invention can alsobe modified by altering the order or composition of certain residues, itbeing readily appreciated that the core immuostimulatory sequence,X₁LYQYMDDV (SEQ ID NO:1), may generally not be altered without anadverse effect on biological activity. The noncritical amino acids neednot be limited to those naturally occurring in proteins, such asL-α-amino acids, or their D-isomers, but may include non-natural aminoacids as well, such as β-γ-δ-amino acids, as well as many derivatives ofL-α-amino acids.

Fusion molecules may also comprise isosteres of two or more residues inthe immunogenic peptide. An isostere as defined here is a sequence oftwo or more residues that can be substituted for a second sequencebecause the steric conformation of the first sequence fits a bindingsite specific for the second sequence. The term specifically includespeptide backbone modifications well known to those skilled in the art.Such modifications include modifications of the amide nitrogen, theα-carbon, amide carbonyl, complete replacement of the amide bond,extensions, deletions or backbone crosslinks. See, generally, Spatola,Chemistry and Biochemistry of Amino Acids, peptides and Proteins, Vol.VII (Weinstein ed., 1983).

Modifications of fusion molecules with various amino acid mimetics orunnatural amino acids are particularly useful in increasing thestability of the peptide in vivo. Stability can be assayed in a numberof ways. For instance, peptidases and various biological media, such ashuman plasma and serum, have been used to test stability. See, e.g.,Verhoef et al, Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Halflife of the peptides of the present invention is conveniently determinedusing a 25% human serum (v/v) assay. The protocol is generally asfollows. Pooled human serum (Type AB, non-heat inactivated) isdelipidated by centrifugation before use. The serum is then diluted to25% with RPMI tissue culture media and used to test peptide stability.At predetermined time intervals a small amount of reaction solution isremoved and added to either 6% aqueous trichloracetic acid or ethanol.The cloudy reaction sample is cooled (4° C.) for 15 minutes and thenspun to pellet the precipitated serum proteins. The presence of thepeptides is then determined by reversed-phase HPLC usingstability-specific chromatography conditions. The peptides of thepresent invention may be modified to provide desired attributes otherthan improved serum half life. For example, the ability of the peptidesto induce CTL activity can be enhanced by linkage to a sequence thatcontains at least one epitope capable of inducing a T helper cellresponse. Particularly preferred immunostimulatory peptides/T helperconjugates are linked together by a spacer molecule. The spacer istypically comprised of relatively small, neutral molecules, such asamino acids or amino acid mimetics, which are substantially unchargedunder physiological conditions. The spacers are typically selected from,e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids orneutral polar amino acids. It will be understood that the optionallypresent spacer need not be comprised of the same residues and thus maybe a hetero- or homo-oligomer. When present, the spacer will usually beat least one or two residues, more usually three to six residues.Alternatively, the immunostimulatory peptide may be linked to the Thelper peptide without a spacer. Linkage to the T helper peptide may beat the amino or carboxy terminus of the immunostimulatory peptide. Theamino terminus of either the immunostimulatory peptide or the T helperpeptide may be acylated. The carbotyl terminus of either theimmunostimulatory peptide or the T helper peptide may also be modified,e.g., by amidation, esterfication or reduction of the carboxyl group.Methods for performing these modifications are well known to those ofskill in the art.

In some embodiments of the invention it may, for example, be desirableto include in the pharmaceutical compositions of the invention at leastone component which assists in priming a CTL response. Lipids have beenidentified as agents capable of assisting the priming CTL in vivoagainst viral antigens. For example, palmitic acid residues can beattached to the alpha and epsilon amino groups of a Lys residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunostimulatory peptide. Thelipidated peptide can then be injected directly in a micellar form,incorporated into a liposome or emulsified in an adjuvant, e.g.,incomplete Freund's adjuvant. In a preferred embodiment a particularlyeffective immunogen comprises palmitic acid attached to alpha andepsilon amino groups of Lys, which is attached via linkage, e.g.,Ser-Ser, to the amino terminus of the immunostimulatory peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS) can be used to prime virus specific CTL when covalently attached toan appropriate peptide. See, Deres et al., Nature 342:561-564 (1989),incorporated herein by reference. Immunostimulatory peptides of theinvention can be coupled to P₃ CSS, for example, and the lipopeptideadministered to an individual to specifically prime a CTL response tothe target antigen. Further, as the induction of neutralizing antibodiescan also be primed with P₃ CSS conjugated to a peptide that displays anappropriate epitope, the two compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses toinfection.

The peptides of this invention are thought to have utility for a vaccineto prevent HCV infection or for therapeutic purposes in individualsinfected with HCV. For example, the peptides can be used by themselves,or they can be used to prepare immunogenic conjugates in which a peptideis conjugated to an agent which provokes an immune response to a complexcomprising the conjugated peptide bound to a carrier protein, accordingto methods known in the art. See, for instance, M. F. Good, Science235:1059-1062 (1987); and Palker, T. J., J. Imm. 142:3612-3619 (1989).Agents which can be conjugated to peptides to provoke an immune responseinclude toxoids such as diphtheria toxoid or tetanus toxoids, which arecommonly recognized by the body (of immunized persons) and eliminated bythe immune system. Alternatively, a gene sequence encoding the peptidemay be incorporated into a recombinant gene and expressed as part of avector, for instance, a recombinant virus such as vaccinia virus made bythe method of Chakrabarti, S., et al., Nature 320:535-537 (1986).

The peptide of the present invention also may be incorporated into alarger peptide comprising additional epitopes, either other T cellepitopes or B cell epitopes. Thus, the peptide may be used as part of amultivalent vaccine which induces cytotoxic T cell responses to multipleepitopes of HCV or of HCV and another virus. In addition, themultivalent vaccine peptide may include helper T cell epitopes and Bcell epitopes of HCV or another virus, to effect induction of anantibody response as well as a cytotoxic T cell response. For instance,one could attach a helper T cell epitope from HIV, such as thosedescribed in Cease K. B., et al., Proc. Natl. Acad. Sci. USA84:4249-4253 (1987), to provide T cell help for the CTL response. Alsosee Berzofsky, J. A., et al., J. Clin. Invest. 88:876-884 (1991); forpeptides generating antiviral cytotoxic T lymphocytes, Hart, M. K., etal., Proc Natl Acad Sci USA 88:9448-9452 (1991); and for peptidesinducing an antibody response, Hart M., K., et al., J. Immunol.145:2677-2685 (1990). Collett, N. S., V. Moennig, and M. C. Horzinek.1989. Recent advances in pestivirus research. J. Gen. Virol. 70:253-266.

Those skilled in the art of preparing pharmaceutical compositions willrealize how to prepare the peptides and conjugates described above forpharmaceutical use in composition comprising accepted pharmaceuticalcarriers.

IV. Methods for Assessing an Immune Response Against HIV-1

The peptides also find use as diagnostic reagents. For example, apeptide of the invention may be used to determine the susceptibility ofa particular individual to a treatment regimen which employs the peptideor related peptides, and thus may be helpful in modifying an existingtreatment protocol or in determining a prognosis for an affectedindividual. In addition, the peptides may also be used to predict whichindividuals will be at substantial risk for developing chronicinfection.

An important aspect to the diagnosis and treatment of HIV-1 is adetermination of the presence of viral infection, and when infection ispresent, monitoring the viral load or the infected individual. Thepresent invention addresses these issues by providing methods ofassessing immune function or diagnosing exposure to HIV-1 for a subject.Performing the methods involves contacting a blood sample from thesubject that contains T cells with an immunostimulatory peptide of thepresent invention; and, determining if peptide contact induces an immuneresponse, preferably a CTL response. The blood sample will need tocontain antigen-presenting cells. These cells may be endogenous to thesample, or added from an external source. Antigen-presenting cells canbe normal cells such as peripheral blood mononuclear cells or dendriticcells (Inaba, et al., J. Exp. Med. 166:182 (1987); and Boog, Eur. J.Immunol. 18:219 [1988]).

Next, peptides that test positive in the MHC class I binding assay areassayed for the ability of the peptides to induce specific CTL responsesin vitro. For instance, antigen-presenting cells that have beenincubated with a peptide can be assayed for the ability to induce CTLresponses in responder cell populations.

Alternatively, mutant mammalian cell lines that are deficient in theirability to load class I molecules with internally processed peptides,such as the mouse cell lines RMA-S (Karre, et al. Nature, 319:675(1986); Ljunggren, et al., Eur. J. Immunol. 21:2963-2970 (1991)), andthe human somatic T cell hybridoma, T-2 (Cerundolo, et al., Nature345:449-452 (1990)) and that have been transfected with the appropriatehuman class I genes may be conveniently used. To test for the capacityof an immunostimulatory peptide of the invention to induce in vitroprimary CTL response, the peptide is added to the cells. Othereukaryotic cell lines which could be used include various insect celllines such as mosquito larvae (ATCC cell lines CCL 125, 126, 1660, 1591,6585, 6586), silkworm (ATTC CRL 8851), armyworm (ATCC CRL 1711), moth(ATCC CCL 80) and Drosophila cell lines such as a Schneider cell line(see Schneider J. Embryol. Exp. Morphol. 27:353-365 [1927]). That havebeen transfected with the appropriate human class I MHC allele encodinggenes and the human B₂ microglobulin genes.

Alternatively, IFN-γ and/or RANTES production by stimulated T cells canbe measured in the T cell culture supernatant. Methods for measuring CTLresponse, RANTES and IFN-γ production of stimulated T cells are wellknown in the art, some of which are discussed in the general methods ofthe examples section, below and elsewhere in this specification.

The immunogenic peptides of this invention may also be used to makemonoclonal antibodies. Such antibodies may be useful as potentialdiagnostic or therapeutic agents.

V. Vaccines for Immunizing Against HIV-1

The peptides of the present invention and pharmaceutical and vaccinecompositions thereof are useful for administration to mammals,particularly humans, to treat and/or prevent viral infection and cancer.Examples of diseases which can be treated using the immunogenic peptidesof the invention include indications of the presence of the HIV-1 virusin an individual including asymptomatic seropositivity, aids-relatedcomplex (arc), and acquired immunodeficiency syndrome (AIDS).

Pharmaceutical compositions of the immunostimulatory peptides of theinvention are administered to an individual already suffering fromindications of the presence of the HIV-1 virus including asymptomaticseropositivity, aids-related complex (arc), and acquiredimmunodeficiency syndrome (AIDS). Those in the incubation phase or theacute phase of infection can be treated with the immunogenic peptidesseparately or in conjunction with other treatments, as appropriate. Intherapeutic applications, compositions are administered to a patient inan amount sufficient to elicit an effective immune response, preferablya CTL response to the virus and cure, or at least partially arrestsymptoms and/or complications. An amount adequate to accomplish this isdefined as “therapeutically effective dose.” Amounts effective for thisuse will depend on, e.g., the peptide and/or protein composition, themanner of administration, the stage and severity of the disease beingtreated, the weight and general state of health of the patient, and thejudgment of the prescribing physician, but generally range for theinitial immunization (that is for therapeutic or prophylacticadministration) from about 0.001 to about 200 mg/kg, more preferablyabout 0.01 to about 100 mg/kg, most preferably about 0.1 to 50 mg/kgpeptide, followed by boosting dosages of from about 0.001 to about 100mg/kg, more preferably about 0.01 to about 50 mg/kg peptide pursuant toa boosting regimen over weeks to months, depending upon the patient'sresponse and condition determined by measuring specific CTL activity inthe patient's blood as described previously and in the examples thatfollow.

It should be kept in mind that the peptides and compositions of thepresent invention may generally be employed individuals with chronic HIVinfections, that is, life-threatening or potentially life threateningsituations. In such cases, in view of the minimization of extraneoussubstances and the relative nontoxic nature of the peptides, it ispossible and may be felt desirable by the treating physician toadminister substantial excesses of these peptide compositions.

For therapeutic use, administration should begin at the first sign ofviral infection. This is followed by boosting doses until at leastsymptoms are substantially abated and for a period thereafter. Loadingdoses followed by boosting doses may be required.

Treatment of an infected individual with the compositions of theinvention may hasten resolution of the infection in acutely infectedindividuals. For those individuals susceptible (or predisposed) todeveloping chronic infection the compositions are particularly useful inmethods for preventing the infection. Where susceptible individuals areidentified prior to or during infection the composition can be targetedto them, minimizing need for administration to a larger population.

The peptide compositions may also be used to stimulate the immune systemto eliminate virus-infected cells in carriers. It is important toprovide an amount of immuno-potentiating peptide in a formulation andmode of administration sufficient to effectively stimulate a cytotoxic Tcell response. Thus, in these cases, a representative dose is in therange of about 0.001 to about 200 mg/kg, more preferably about 0.01 toabout 100 mg/kg, most preferably about 0.1 to 50 mg/kg peptide per dose.Immunizing doses followed by boosting doses at established intervals,e.g., from one to four weeks, may be required, possibly for a prolongedperiod of time to effectively immunize an individual. Administrationshould continue until at least clinical symptoms or laboratory testsindicate that the viral infection has been eliminated or substantiallyabated and for a period thereafter.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral or local administration. Preferably, thepharmaceutical compositions are administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin a pharmaceutically acceptable excipient, preferably an aqueouscarrier. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.These compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of immunostimulatory peptides of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

Both peptides and the nucleic acids encoding them of the invention mayalso be administered via liposomes. Liposomes are useful in increasingthe half-life of the peptides. Liposomes include emulsions, foams,micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In liposome preparations thepeptide to be delivered may be incorporated as part of a liposome, aloneor in conjunction with a molecule that binds to, e.g., a receptorprevalent among lymphoid cells, such as monoclonal antibodies that bindto the CD45 antigen, or with other therapeutic or immunogeniccompositions. Thus, liposomes filled with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the selected therapeutic/immunogenic peptidecompositions.

Liposomes for use in the invention are formed from standardvesicle-forming lipids, which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of, e.g., liposome size,acid lability and stability of the liposomes in the blood stream. Avariety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporatedherein by reference.

For targeting to immune cells, a ligand to be incorporated into theliposome can include, e.g., antibodies or fragments thereof specific forcell surface determinants of the desired immune system cells. A liposomesuspension containing an immunostimulatory peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedincluding, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally about 10% to about 95%of active ingredient, that is, one or more peptides of the invention,and more preferably at a concentration of about 25% to about 75%.

For aerosol administration, the immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are 0.01%-20% by weight, preferably1%-10%. The surfactant must be nontoxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olesteric and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride. Mixed esters, such as mixed or natural glycerides maybe employed. The surfactant may constitute 0.1%-20% by weight of thecomposition, preferably 0.25-5%. The balance of the composition isordinarily propellant. A carrier can also be included, as desired, aswith, e.g., lecithin for intranasal delivery.

Another aspect the present invention is directed to vaccines thatcontain as an active ingredient an immunogenically effective amount ofan immunostimulatory peptide as described herein. The peptide(s) may beintroduced into a host, including humans, linked to its own carrier oras a homopolymer or heteropolymer of active peptide units. Such apolymer has the advantage of increased immunological reaction and, wheredifferent peptides are used to make up the polymer, the additionalability to induce antibodies and/or CTLs that react with differentantigenic determinants of HIV virus. Useful carriers are well known inthe art, and include, e.g., thyroglobulin, albumins such as bovine serumalbumin, tetanus toxoid, polyamino acids such as poly(lysine:glutamicacid), hepatitis B virus core protein, hepatitis B virus recombinantvaccine and the like. The vaccines can also contain a physiologicallytolerable (acceptable) diluent such as water, phosphate buffered saline,or saline, and further typically include an adjuvant. Adjuvants such asincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, oralum are materials well known in the art. As mentioned above, CTLresponses can be primed by conjugating peptides of the invention tolipids, such as P₃ CSS. Upon immunization with a peptide composition asdescribed herein, via injection, aerosol, oral, transdermal or otherroute, the immune system of the host responds to the vaccine byproducing large amounts of CTLs specific for the desired antigen, andthe host becomes at least partially immune to later infection, orresistant to developing chronic infection.

Vaccine compositions containing the peptides, and nucleic acids encodingthem, of the invention are administered to a patient susceptible to orotherwise at risk of HIV infection, to elicit an immune response againstthe antigen and thus enhance the patient's own immune responsecapabilities. Such an amount is defined to be an “immunogenicallyeffective dose.” In this use, the precise amounts again depend on thepatient's state of health and weight, the mode of administration, thenature of the formulation, etc., but generally range from about 0.001 toabout 200 mg/kg, more preferably about 0.01 to about 100 mg/kg, mostpreferably about 0.1 to 50 mg/kg peptide, more commonly from about 0.01to about 100 mg/kg, more preferably about 0.1 to 50 mg/kg peptide/bodyweight.

In some instances it may be desirable to combine the peptide vaccines ofthe invention with vaccines which induce neutralizing antibody responsesto HIV-1, particularly to viral envelope antigens.

Immunostimulatory peptides may also be used to elicit CTL ex vivo, aswell. The resulting CTL, can be used to treat patients that do notrespond to other conventional forms of therapy, or will not respond to apeptide vaccine approach of therapy. See, e.g., U.S. Pat. No. 6,037,135for methods of performing ex vivo CTL therapy. Methods of re-introducingcellular components are known in the art and include procedures such asthose exemplified in U.S. Pat. No. 4,844,893 to Honsik, et al. and U.S.Pat. No. 4,690,915 to Rosenberg. For example, administration ofactivated CD8+ cells via intravenous infusion is appropriate.

Peptides and proteins of the present invention may also be used to pulseautologous dendritic cells as a means of immunization against thepeptide.

Live Vaccines

For therapeutic or immunization purposes, the peptides of the inventioncan also be expressed by attenuated viral hosts, such as vaccinia orfowlpox. This approach involves the use of the virus as a vector toexpress nucleotide sequences that encode the peptides of the invention.Upon introduction into an infected host or uninfected host, therecombinant virus expresses the immunogenic peptide, and thereby elicitsa host CTL response. Vaccinia vectors and methods useful in immunizationprotocols are described in, e.g., U.S. Pat. No. 4,722,848, incorporatedherein by reference. Another vector is BCG (Bacille Calmette Guerin).BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)).A wide variety of other vectors useful for therapeutic administration orimmunization of the peptides of the invention, e.g., Salmonella typhivectors and the like, will be apparent to those skilled in the art fromthe description herein.

Gene Therapy

Delivery into a patient of nucleic acids encoding peptides and proteinsof the present invention may be either direct, in which case the patientis directly exposed to the nucleic acid or nucleic acid-carryingvectors, or indirect, in which case, cells are first transformed withthe nucleic acids in vitro, then transplanted into the patient. Thesetwo approaches are known, respectively, as in vivo or ex vivo genetherapy.

For example, the nucleic acid sequences may directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing them as part of an appropriate nucleic acid expressionvector and administering it so that they become intracellular, e.g., byinfection using defective or attenuated retrovirals or other viralvectors (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering them in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol.Chem. 262:4429-4432 (1987)) (which can be used to target cell typesspecifically expressing the receptors), etc. In another embodiment,nucleic acid-ligand complexes can be formed in which the ligandcomprises a fusogenic viral peptide to disrupt endosomes, allowing thenucleic acid to avoid lysosomal degradation. In yet another embodiment,the nucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180; WO 92/22635; WO092/20316; WO 93/14188, WO93/20221). Alternatively, the nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); Zijlstra et al, Nature 342:435-438 (1989)).

Nucleic acids of the present invention may also serve as effectivevaccines, by introducing them into suitable cells where they will beexpressed and either secreted, or displayed on the cell surface of thetransformed cell. For example nucleic acids encoding peptides andproteins of the present invention them may be used to transducedendritic cells, which in turn can be used as vaccines for immunization.

Other modes of gene therapy are also contemplated by the presentinvention. For general reviews of the methods of gene therapy, seeGoldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan andAnderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);and Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY (1990).

Although the foregoing invention has been described in some detail byway of illustration and example for clarity and understanding, it willbe readily apparent to one of ordinary skill in the art in light of theteachings of this invention that certain changes and modifications maybe made thereto without departing from the spirit and scope of theappended claims.

EXAMPLES

As can be appreciated from the disclosure provided above, the presentinvention has a wide variety of applications. Accordingly, the followingexamples are offered for illustration purposes and are not intended tobe construed as a limitation on the invention in any way. Those of skillin the art will readily recognize a variety of noncritical parametersthat could be changed or modified to yield essentially similar results.

General Methods

The following methods are general to all examples that follow. Theinventors wish to thank Dr. Linda Sherman of the Scripps ResearchInstitute, La Jolla, Calif., for kindly donating the Jurkat-A2K^(b) cellline, A2Kb mice. The inventors would also like to thank Dr. BernardMoss, NIAID, for his gift of HIV reverse transcriptase (vCF21) orβ-galactosidase (vSC8) and Dr. Victor Engelhard of the University ofVirginia for his donation of a C1R.AAD cell line.

Synthetic Peptides

Peptides were prepared in an automated multiple peptide synthesizer(Symphony; Protein Technologies, Inc.) using Fmoc chemistry. Peptideswere purified by reverse-phase HPLC, and their sequences confirmed on anautomated sequencer (477A; Applied Biosystems, Foster City, Calif.).Some peptides were purchased from Multiple Peptide Systems (San Diego,Calif.).

Cells

A Jurkat-A2K^(b) cell line was transfected with an HLA chimeric moleculecontaining α1 and α2 domains from human HLA-A2.1 and an α3 domain frommouse H-2K^(b). A C1R.AAD cell line (HMYC1R transfected with an HLAchimeric molecule containing α1 and α2 domains from human HLA-A2.1 andan α3 domain from mouse H-2D^(d)) (Sarobe, P. et al., J. Clin. Invest.102:1239). Cell lines were maintained in 10% FCS RPMI containing 1 mMsodium pyruvate, nonessential amino acids (Biofluid, Rockville, Md.), 4mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μM2-mercaptoethanol.

Mice

Transgenic A2Kb mice (Vitiello, et al., J. Exp. Med. 173:1007) andtransgenic HHD-2 mice (Pascolo, et al., J. Exp. Med. 185:2043; andFirat, H., S., et al., Eur J Immunol 31:3064.) were bred in our colonyat BioCon Inc. (Rockville, Md.). A2Kb mice express a chimeric HLA-A2.1molecule with the α3 domain derived from the murine H-2K^(b). HHD-2 micehave murine β2-microglobulin and murine H-2D^(b) genes knocked out.HHD-2 mice are also transgenic for a human HLA-A2.1 that has acovalently-linked human β2-microglobulin and a murine D^(b)-derived α3domain, which allows interaction with mouse CD8. Because of thesegenetic alterations, the only class I MHC molecule expressed by HHD-2mice is human HLA-A2.1. The genetic changes to both mice allow forbetter binding of murine CD8. Both strains are on a C57BL/6 background.

T2 Binding Assay

Peptide binding to HLA molecules was measured using the T2 mutant cellline as described (Sarobe, P. et al., J. Clin. Invest. 102:1239; andNijman, H. W., J., et al., Eur. J. Immunol. 23:1215.). Briefly, T2 cells(3×10⁵/well) were incubated overnight in 96-well plates with culturemedium (a 1:1 mixture of RPMI 1640/EHAA containing 2.5% FBS, 100 U/mlpenicillin, 100 μg/ml streptomycin) with 10 μg/ml human β2-microglobulin(Sigma Chemical Co., St. Louis, Mo.) and different peptideconcentrations. The next day, cells were washed twice with cold PBScontaining 2% FBS and incubated for 30 min at 4° C. with anti-HLA-A2.1BB7.2 mAb (1/100 dilution of hybridoma supernatant) and 5 μg/mlFITC-labelled goat anti-mouse Ig (Pharmingen, San Diego, Calif.). Cellswere washed twice after each incubation, and HLA-A2.1 expression wasmeasured by flow cytometry (FACScan; Becton Dickinson, Mountain View,Calif.). HLA-A2.1 expression was quantified by fluorescence index (FI)according to the formula: [FI=(mean fluorescence with peptide−meanfluorescence without peptide)/mean fluorescence without peptide.Background fluorescence without BB7.2 was subtracted for each individualvalue]. To compare the different peptides, FI_(0.5), the peptideconcentration that increases HLA-A2.1 expression by 50% over no peptidecontrol background, was calculated from the titration curve for eachpeptide.

FIG. 1A is a comparison of the HLA-A2 binding curves among the wild typeRT (179-187), VIYQYMDDL (SEQ ID NO:7), RT-1Y (YIYQYMDDL; SEQ ID NO:8),RT-2L9V (VLYQYMDDV; SEQ ID NO:2), and RT-1Y2L9V (YLYQYMDDV: SEQ ID NO:3)in the T2-binding assay.

FIG. 1B compares HLA-A2 binding curves among the RT-2L9V, p17-WT(SLYNTVATL; SEQ ID NO:9), RT-1Y2L9V and FMP (GILGFVFTL; SEQ ID NO:10).

CTL Generation in A2Kb and HHD-2 Transgenic Mice

Mice more than 8-week-old were immunized subcutaneously at the base ofthe tail with 100 μl of an emulsion containing 1:1 incomplete Freund'sadjuvant (IFA) and PBS solution with antigens and cytokines (50 nmol CTLepitope, 50 nmol HBV core 128-140 helper epitope, 5 μg of IL-12, and 5μg of granulocyte macrophage colony stimulating factor (GM-CSF)). Micewere boosted 2 wk later, and spleens removed 10-14 days after the boost.Immune spleen cells (2.5×10⁶/well) were stimulated in 24-well plateswith autologous spleen cells (5×10⁶/well) pulsed for 2 h with 10 μM CTLepitope peptide in CTM with 10% T-Stim (Collaborative BiochemicalProducts, Bedford, Mass.). After more than 4 in vitro stimulations withpeptide-pulsed syngeneic spleen cells, CTL lines were maintained byweekly restimulation of 1×10⁶ CTL/well with 4×10⁶ peptide pulsedirradiated (3,300 rads) syngeneic spleen cells as feeders, or by weeklystimulation of 1×10⁶ CTL/well with 3.8×10⁶ peptide pulsed irradiatedC57BL/6 spleen cells and 1−3×10⁵ peptide pulsed and irradiated (15,000rad) Jurkat-A2K^(b) transfectant cells.

Cytotoxicity Assay

CTL activity was measured using a 4-h assay with ⁵¹Cr-labeled targetcells. Target cells (10⁶) were pulsed in 100 μl CTM and 150 μCi⁵¹Cr for1.5 h, washed three times, and added at 3,000 cells/well to the 96-wellround-bottom plates with different peptide concentrations. Effectorcells were added 2 h later, and the supernatants were harvested andcounted after an additional 4 h of incubation. The percentage ofspecific ⁵¹Cr release was calculated as 100×(experimentalrelease−spontaneous release)/(Maximum release−spontaneous release).Spontaneous release was determined from target cells incubated withouteffector cells, and maximum release was determined in the presence of0.1 M HCl. Jurkat-A2K^(b) lines or C1R.AAD cell lines were used astargets.

IFN-γ and RANTES Assay

IFN-γ and RANTES in the culture supernatant were determined by ELISA kit(R&D, Minneapolis, Mass.) according to the manufacturer's instructions.All samples were analyzed in triplicate.

Protection Assay from Viral Challenge

Female mice were immunized with the same protocol as in the CTLgeneration protocol described above, boosted i.p. 2 weeks after primaryimmunization, and challenged i.p. 30 days later with recombinantvaccinia virus (2×10⁷ pfu/mouse) expressing HIV reverse transcriptase(vCF21) or β-galactosidase (vSC8). Five days later virus titers in theovaries of individual mice were determined on BSC-1 indicator cells aspreviously described (Ahlers, J. D., et al., Int Immunol 13:897).

Example 1 RT Ala-Substituted Peptides Binding to HLA-A2.1 Molecules

This example is designed to determine which residues, other than theanchor residues 2 and 9, are important in RT-WT peptide binding to MHCmolecules.

Binding affinity of wild type RT (179-187) (RT-WT) using the T2 bindingassay, measuring the cell surface stabilization of HLA-A2.1 moleculesafter incubation with peptide. Relative affinity for MHC molecules wasdetermined for each peptide in Table 2 by comparing their FI_(0.5)values as calculated from titration curves against HLA-A2 molecules.Using this method, an FI_(0.5) of 41.9 μsM was calculated for RT-WT.This binding affinity was much weaker than that of other 9-mer peptidestested in our lab such as hepatitis C virus peptide C7A2 (Sarobe, P., etal., (1998) J. Clin. Invest. 102.1239), Flu matrix peptide 58-66 (FMP)(Gotch, F. M., et al., (1987). Nature 326. 881.), and HIV-gag peptideSLYNTVATL (SEQ ID NO:9) (McMichael, A. J., and B. D. Walker. (1994),AIDS 8 (suppl 1):S155; See also Table 2). In a set of experiments todefine key functional residues, peptides with alanine substitutions ateach one of the positions were synthesized and tested in binding assays,as described above. The results of these experiments are summarized inTable 2, below.

TABLE 2 Binding of RT (179-187)-wild type and- substituted peptide toHLA-A2. SEQ Peptide Sequence ID NO: FI_(0.5) RT VIYQYMDDL 7 41.9(179-187)-WT 1A AIYQYMDDL 12 33.7 2A VAYQYMDDL 13 >100 3A VIAQYMDDL 1441.2 4A VIYAYMDDL 15 40.7 5A VIYQAMDDL 16 95.6 6A VIYQYADDL 17 17.4 7AVIYQYMADL 18 >100 8A VIYQYMDAL 19 35.9 9A VIYQYMDDA 20 57.9 2L VLYQYMDDL21 19.2 9V VIYQYMDDV 22 19.9 2L9V VLYQYMDDV 2 5.7 (RT-2L9V) gag (p17)SLYNTVATL 9 2.21 (77-85) Flu-MP GILGFVFTL 10 0.24 (58-66)

As indicated in table 2, alanine substitutions at the 2 and 7 positionscaused almost complete loss of binding to HLA-A2. Alanine substitutionat position 5 also caused a substantial decrease in binding, whereas amoderate decrease in binding was observed when alanine was substitutedfor leucine at position 9. These data suggest that, in addition to theanchor residues 2 and 9, the amino acid residue at positions 5 and 7 arealso important to peptide binding to HLA-A2.

Example 2 Leu and/or Val-Substitution at Anchor Regions in RT-WT

In an attempt to enhance peptide binding to MHC molecules, peptides withsubstitutions of leucine and/or valine, at the anchor positions 2 and 9respectively, were synthesized and tested in the binding assay, asdescribed above. The leucine and valine substitutions were chosenbecause these are the amino acids that predominate at the respectivepositions in peptides known to bind HLA-A2.1 molecules (Rammensee,H.-G., et al., (1995) Immunogenetics 41:178). Peptides substituted withleucine at position 2 or valine at position 9 had around 2-fold higheraffinity for HLA-A2 than RT-WT. However, a peptide substituted at bothpositions, RT-2L9V, had around an 8-fold higher binding affinity forHLA-A2 than RT-WT. This affinity was higher than that of any otheralanine-substituted peptides of RT (179-187) tested. (see table 2).

Example 3 Comparison of the Binding Affinity Between Substitutions inAnchor Region and Tyrosine-Substitution in Position 1

Recent studies reported that a Tyrosine substitution in the firstposition (P¹Y) can increase peptide/MHC binding without alteringantigenic specificity (Pogue, R. R., et al., (1995) Proc. Natl. Acad.Sci. U.S.A. 92:8 166; Tourdot, S., A. et al., (2000) Eur J Immunol30:3411). Based on these studies, we used the T2 binding assay tocompare peptide/MHC binding among 4 derivative peptides:

RT-WT, RT-2L9V, RT-1Y (YIYQYMDDL); SEQ ID NO: 8), and RT-1Y2L9V(YLYQYMDDV); SEQ ID NO: 3)

As shown in FIG. 1A, RT-2L9V displayed much better binding than theRT-1Y, while both substituted peptides had higher affinity than RT-WT.RT-1Y2L9V displayed the highest affinity of all the peptides. Thebinding ability of RT-1Y2L9V was almost as good as that of FMP (FIG.1B). This data suggests that RT-2L9V and RT-1Y2L9V were the bestcandidates for an epitope enhanced peptide.

Example 4 Recognition of RT-Variant Peptides by CTL Lines from A2K^(b)-and HHD-2-Transgenic Mice

To determine residues involved in CTL recognition, we immunized HLA-A2transgenic mice, using two different strains.

RT-WT and -2L9V specific CTL lines were separately developed from bothA2Kb and HHD-2 transgenic mice, and an RT-1Y2L9V specific CTL line wasdeveloped from HHD-2 mice by immunizing the mice with the respectivepeptides, followed by several rounds (e.g., 5 rounds) of stimulationwith each respective peptide. The resulting CTL lines had almostcompletely non-overlapping Vβ repertoires. (In the case of A2Kb mice,the RT-WT specific CTL line had Vβ2, 3 and 12 while the RT-2L9V specificCTL line had Vβ3, 4, 5, 8 and 10b. In the case of HHD-2 mice, the RT-WTspecific CTL had Vβ8.1 or 8.2 while the RT-2L9V specific CTL had Vβ4,data not shown.)

Crossreactivity among RT-WT, RT-2L9V and RT-1Y2L9V was checked using thepeptide-specific CTL lines (FIG. 2). Jurkat-A2K^(b) transfectant cellsor C1R.AAD cells were used as a target. RT-WT, RT-2L9V and RT-1Y2L9Vspecific CTL lines killed target cells pulsed with an adequate antigenconcentration in an antigen-specific manner. The peptide-specific CTLlines were crossreactive with targets pulsed with the wild type peptide.In the case of A2Kb-derived CTL lines, RT-2L9V coated targets werekilled at lower a concentration than RT-WT-coated targets, consistentwith the higher affinity of the RT-2L9V peptide and the crossreactivityof the CTL lines for the two peptides. Unexpectedly, theRT-2L9V-specific CTL line killed RT-WT pulsed targets at more than onelog lower peptide concentration than did the line raised against thispeptide, indicating that the RT-2L9V peptide also elicited higheravidity CTL.

All 3 peptide-specific HHD-2-derived CTL lines recognized wildtype-pulsed targets. RT-2L9V-specific CTL recognized the wild typepeptide-pulsed targets to the same extent as the RT-WT specific CTL.However, 1Y2L9V-specific CTL recognition of the wild type peptide-pulsedtargets was paradoxically weaker than that by the other two CTL lines(FIG. 2B). Moreover, RT-WT specific CTL did not recognize RT-1Y andRT-1Y2L9V-pulsed targets (FIG. 2B). Furthermore, RT-1Y2L9V-specific CTLrecognized targets pulsed with peptides having a tyrosine substitutionin position 1 preferentially over peptides not mutated in position 1,even though RT-1Y has lower binding affinity to the HLA-A2 molecule thanRT-2L9V in the T2-binding assay.

These data indicate that the difference between V and Y at position 1can clearly be distinguished by the T cell receptor, and T-cellspecificity for V over Y can override the effect of the higher affinityfor MHC shown by the peptides substituted at position 1 with tyrosine.This indicates that the amino acid in position 1 of anHLA-A2.1-restricted CD8 epitope could be critical to correct recognitionby the T cell receptor, in contrast to the examples disclosed inTourdot, S., A. et al., (2000) Eur J Immunol 30:3411.

In addition, the data indicate that RT-2L9V-specific CTLs derived fromHLA-A2 transgenic mice have the same or higher avidity for targetspulsed with RT-WT, when compared to RT-WT specific CTLs.

FIG. 2 is a comparison of antigenic potency by RT-WT, -2L9V and -1Y2L9VCTL lines. FIG. 2A illustrates recognition of RT-WT and RT-2L9V peptidesby RT-WT and RT-2L9V specific CTL lines from A2Kb-transgenic mice as afunction of peptide concentration reveals difference in peptide affinityfor HLA-A2 and CTL avidity for the same peptide-MHC complexes. (E/Tratio, 10:1). FIG. 2B depicts the recognition of RT-WT, -1Y, -2L9V and-1Y2L9V peptides by RT-WT, -2L9V and -1Y2L9V specific CTL lines fromHHD-2-transgenic mice as a function of peptide concentration revealsdifference in peptide affinity for HLA-A2 and CTL avidity for the samepeptide-MHC complexes. (E/T ratio, 10:1)

Example 5 IFN-γ and RANTES Production from the RT-Specific CTL LinesStimulated by RT-Variant Peptides

This example describes tests of the peptide-specific IFN-γ and RANTESproduction by each peptide-specific CTL line described above, as afunction of peptide concentration. These tests allow us to compareinducibility by the RT-WT and substituted peptides of other forms of Tcell activity important to the cellular immune response (see FIGS. 3 and4).

IFN-γ is known to contribute to clearance of recombinant vaccinia virusin mice (Harris, N., R. M. Buller, and G. Karupiah, (1995). J Virol.69:910.). RANTES can inhibit binding of HIV to its coreceptor, CCR5(Cocchi, F., A. L. et al., (1995) Science 270:1811). In A2Kb mice, theRT-2L9V peptide induced more IFN-γ production by the RT-WT specific CTLline than the RT-WT peptide itself, as the peptide concentration wasreduced. The CTL raised against RT-2L9V also appeared to have higheravidity for the RT-WT peptide than the CTL raised against the wild typepeptide itself, in that the titration curve for RT-WT was shifted andthe difference in the IFN-γ production by these two CTL lines from A2Kbmice induced by the same RT-WT peptide was more than 100-fold at 0.1 μMof peptide (FIG. 3A). The RT-2L9V peptide also could induce more IFN-γproduction by the RT-2L9V specific CTL line than the RT-WT peptide.

In HHD mice, IFN-γ production from the RT-1Y2L9V specific CTL line wasparallel to the binding ability of the peptide to HLA-A2. However, theantigen-specific IFN-γ production by RT-2L9V specific CTL was betterwhen stimulated with RT-2L9V than with RT-1Y2L9V. The RT-2L9V specificCTL line also produced about 10 times more IFN-γ compared to theRT-1Y2L9V specific CTL when stimulated with the wild type peptide (FIG.3B).

RANTES (Regulated upon Activation, Normal T expressed, and presumablySecreted) is a member of the CC chemokine family of inflammatory andimmunoregulatory chemokines. RANTES is also produced by stimulated CTLand has been shown to inhibit HIV infection of human mononuclear cells(Cocchi, F., A. L. et al., (1995) Science 270:1811). Production of thischemokine by CTL therefore could be one of the parameters for effectoractivity in HIV infection.

As shown in FIGS. 4A and 4B, RANTES was produced by all CTL lines in anantigen-specific manner. In A2Kb mice, RANTES production by RT-WTspecific CTL decreased to background levels at 0.1 μM peptide. Incontrast, RANTES production by RT-2L9V specific CTL remained strong,regardless of which peptide was stimulatory (FIG. 4A). These datasuggest that the RT-2L9V-substituted peptide has higher avidity forwild-type specific CTL than the wild type peptide, whereas thecrossreactivity of RT-1Y2L9V-specific CTL for wild-type peptide issignificantly weaker (FIG. 4B).

FIG. 3 is a comparison of IFN-γ and RANTES production induced by RT-WT,-2L9V and -1Y2L9V peptides. After being pulsed with different peptideconcentrations for 2 h, Jurkat-A2K^(b) cells were irradiated at 15,000rad and plated at a hundred thousand cells/well in 96-well round-bottomplates. Five hundred thousand CTLs were added into each well, and thesupernatants were harvested at 48 h. IFN-γ in the culture supernatantwas determined by ELISA kit according to the manufacturer'sinstructions. All samples were used at 2-640-fold dilution and analyzedin triplicate. FIG. 3A graphically illustrates IFN-γ production by RT-WTand -2L9V specific CTL line derived from A2Kb-transgenic mice. FIG. 3Bshows the IFN-γ production by RT-2L9V and -1Y2L9V specific CTL linederived from HHD-2-transgenic mice.

FIG. 4A compares RANTES production by RT-WT and -2L9V specific CTL linederived from A2Kb-transgenic mice, while FIG. 4B compares RANTESproduction by RT-2L9V and -1Y2L9V specific CTL line derived fromHHD-2-transgenic mice.

Example 6 In Vivo Immunogenicity of RT-2L9V and -1Y2L9V Peptide inHLA-A2 Transgenic Mice

This example compares the in vivo immunogenicity of the RT-RW, RT-2L9V,and the RT-1Y2L9V peptides in the A2Kb and HID-2 transgenic mousemodels. To test the ability of RT-WT and RT-2L9V peptides to induce aCTL immune response against RT-WT in A2Kb mice, different groups ofanimals were immunized with RT-WT or RT-2L9V-substituted peptides inconjunction with a helper epitope and cytokines as described in theGeneral Methods, above. The ability of these peptides to induce animmune response was tested in CTL assays after giving the animalsbooster injections with an adequate concentration of the respectivepeptides (FIG. 5A). Both RT-WT and RT-2L9V peptides induced immuneresponses after stimulation with higher peptide concentrations (10 μMand 0.3 μM respectively), but RT-2L9V induced a more vigorous CTL immuneresponse than RT-WT. Dosing with 0.01 μM RT-WT failed to raise a CTLresponse above background levels. 0.01 μM RT-2L9V however was sufficientto induce a CTL response against a RT-WT-pulsed target. These resultsindicate that RT-2L9V induces a stronger CTL response against aRT-WT-pulsed target than RT-WT. Moreover, surprisingly, the CTL responseinduced by RT-2L9V recognizes RT-WT-pulsed targets better than the CTLresponse induced by RT-WT.

In addition, further tests, using antigen-specific IFN-γ production as amarker, demonstrated that tyrosine substitution at position 1 adverselyaffects CTL induction against RT-WT-pulsed targets, despite eliciting astrong CTL response against targets pulsed with the peptide having theposition 1 tyrosine substitution.

HHD-2 mice were immunized twice with 50 nmol peptide and cytokines inIFA as previously described and immune spleen cells were stimulated withsyngeneic spleen cells that had been pulsed with 10 μM peptide andirradiated. After one week, short-term cultures were stimulated, eachwith one of the three peptides. After 48 hr, IFN-γ production for eachculture was determined by measuring the culture supernatant IFN-γconcentration.

No antigen-specific IFN-γ production could be detected in RT-WTimmunized bulk culture using this system. Surprisingly, both RT-2L9V andRT-1Y2L9V-immunized bulk culture induced much higher IFN-γ production(FIG. 5B). Each culture responded best to the peptide used stimulate it.Moreover, IFN-γ produced by RT-2L9V immunized bulk culture against theRT-WT was about 70-fold higher than that produced by the RT-1Y2L9Vculture, despite RT-1Y2L9V displaying much stronger binding to HLA-A2than RT-2L9V. These data suggest that tyrosine substitution at position1 adversely affects CTL induction against RT-WT-pulsed targets, despiteeliciting a strong CTL response against targets pulsed with the peptidehaving the position 1 tyrosine substitution.

FIG. 5A shows the induction of CTL immune response and comparison of CTLavidity against RT-WT in A2Kb-transgenic mice using different RT peptidevariants. A2Kb-transgenic mice were immunized with 50 nmol CTL epitopeRT-WT or -2L9V plus 50 nmol HBVc 128-140 helper epitope and 5 μg IL-12and GM-CSF in IFA. 2 wk later, they were boosted under the sameconditions, and 10-14 days after the boost, spleen cells were removedand stimulated separately in vitro with 10, 0.3 and 0.01 μM CTLpeptide-pulsed spleen cells. A week after the 2^(nd) stimulation inculture, a cytotoxic assay was performed with each concentration ofRT-WT peptide. FIG. 5B illustrates induction of antigen specific IFN-γproduction by the peptide-specific CTL. HHD-2-transgenic mice wereimmunized with 50 nmol CTL epitope RT-WT, -2L9V or -1T2L9V plus 50 nmolHBVc 128-140 helper epitope and 5 μg IL-12 and GM-CSF in IFA. 2 wklater, they were boosted under the same conditions, and 10-14 days afterthe boost, spleen cells were removed and stimulated separately in vitrowith irradiated spleen cells pulsed with the optimum concentration ofeach peptide. 9 days after the stimulation, the cultured cells wererestimulated with each peptide. Each supernatant was assayed for IFN-γby ELISA 48 hr later.

Example 7 Protection Ability of Epitope Enhanced Peptides In Vivo

To determine the ability of RT-2L9V and RT-1Y2L9V to protect againstviral infection in vivo, different populations of HHD-2 mice (describedabove) were immunized with RT-2L9V and RT-1Y2L9V, respectively. Bothpopulations were then challenged with vaccinia virus (vCF21) expressingRT protein as a surrogate to challenge HIV-1 virus. This surrogateapproach was necessary as the HLA-A2.1 transgenic mice cannot beinfected with HIV-1 itself. HHD-2 mice were specifically selected forthis study because the only class I molecule they express is HLA-A2.1(Pascolo, S., N. et al., (1997) J. Exp. Med. 185:2043), so protectioncannot be mediated by CTL restricted to murine MHC molecules. As acontrol, populations of HHD-2 mice, immunized with RT-2L9V and RT-1Y2L9Vrespectively, were challenged with a second vaccinia strain (vSC8) thatdoes not express RT. Neither population of mice displayed protectionagainst vaccinia infection.

FIG. 6 shows the protection induced by immunization with RT-peptides. Onday 30 after the last immunization, female HHD-2 mice, expressing onlythe human HLA-A2.1 class I molecule and no murine class I molecules,were challenged intraperitoneally with 2×10⁷ pfu of vaccinia virusexpressing a reverse transcriptase protein of HIV (vCF21) or aβ-galactosidase protein (vSC8). Five days later, virus titers in theovaries were determined

FIG. 6 illustrates that, in both protection assays, RT-2L9V-immunizedmice were protected against vCF21 infection, resulting in a 4-5 logreduction in virus titer (Exp. 1) or complete protection (6 logreduction) (Exp. 2) compared to unimmunized control animals (p<0.01). Incontrast, RT-1Y2L9V-immunized mice were only partially protected.

These data confirm that RT-2L9V is more a better vaccine candidate thanthe wild type peptide, RT-WT. RT-2L9V is also shown to be a bettervaccine candidate than RT-1Y2L9V, even though RT-1Y2L9V has much higherbinding affinity to HLA-A2 than RT-2L9V.

To ensure a full description of the invention, all publications andpatent applications cited in this specification are herein incorporatedby reference as if each individual publication or patent applicationwere specifically and individually indicated to be incorporated byreference.

1. An immunostimulating peptide comprising an amino acid sequenceX₁LYQYMDDV (SEQ ID NO:1), wherein X₁ is any hydrophobic amino acid. 2.The immunostimulating peptide of claim 1, wherein the amino acidsequence is VLYQYMDDV (SEQ ID NO:2).
 3. A composition comprising: theimmunostimulating peptide of claim
 1. 4. The composition of claim 3,further comprising an immunostimulant.
 5. An immunostimulating peptideor protein comprising the sequence X₁X₂LYQYMDDVX₃ (SEQ ID NO:4) whereinX₁ is a sequence of amino acid residues of between 0 and 200 residues inlength; X₂ is any hydrophobic amino acid; and X₃ is a sequence of aminoacid residues of between 0 and 200 residues in length.
 6. A peptide orprotein comprising an amino acid sequence X₁LYQYMDDV (SEQ ID NO:1),wherein X₁ is any hydrophobic amino acid.
 7. The peptide or protein ofclaim 6, further comprising an acetylated N-terminus.
 8. The peptide orprotein of claim 6, further comprising a modification to the C-terminus,the modification selected from the group consisting of amidation,esterfication, and reduction of a C-terminal amino acid carboxyl group.9. The immunostimulating peptide of claim 1, wherein the amino acidsequence X₁LYQYMDDV (SEQ ID NO:1) is conjugated to a heterologousmolecule to form a fusion molecule.
 10. The immunostimulating peptide ofclaim 9, wherein the heterologous molecule comprises an amino acidsequence for an HIV-1 viral protein.
 11. The immunostimulating peptideof claim 9, wherein the molecule comprises a glycolipid, a glycoprotein,a lipoprotein, or a nucleoprotein.
 12. The immunostimulating peptide ofclaim 9, further comprising an amino acid sequence for animmunostimulating carrier protein.
 13. The immunostimulating peptide ofclaim 9, wherein the heterologous molecule comprises a T helper peptide.14. The immunostimulating peptide of claim 13, further comprising aspacer molecule linking the immunostimulating peptide to the T helperpeptide.
 15. The immunostimulating peptide of claim 1, wherein theimmunostimulating peptide is prepared synthetically.
 16. Theimmunostimulating peptide of claim 1, wherein the immunostimulatingpeptide is prepared recombinantly.
 17. The immunostimulating peptide ofclaim 1, wherein the amino acid sequence is YLYQYMDDV (SEQ ID NO:3). 18.The immunostimulating peptide of claim 1, pulsed onto a dentritic cell.19. An immunostimulating peptide consisting of an amino acid sequenceX₁LYQYMDDV (SEQ ID NO:1), wherein X₁ is any hydrophobic amino acid. 20.The immunostimulating peptide of claim 19, wherein the amino acidsequence is VLYQYMDDV (SEQ ID NO:2).
 21. The immunostimulating peptideof claim 19, wherein the amino acid sequence is YLYQYMDDV (SEQ ID NO:3).22. The immunostimulating peptide of claim 19, wherein theimmunostimulating peptide is prepared synthetically.
 23. Theimmunostimulating peptide of claim 19, wherein the immunostimulatingpeptide is prepared recombinantly.
 24. The immunostimulating peptide ofclaim 19, pulsed onto a dentritic cell.
 25. The immunostimulatingpeptide or protein according to claim 5, wherein the amino acid sequenceis X₁VLYQYMDDVX₃ (SEQ ID NO:5).
 26. The immunostimulating peptide orprotein according to claim 5, wherein the amino acid sequence isX₁YLYQYMDDVX₃ (SEQ ID NO:6).
 27. A peptide or protein comprising anamino acid sequence X₁X₂YQYMDDVX₃ (SEQ ID NO:23) wherein X₁ is asequence of amino acid residues of between 0 and 200 residues in length;X₂ is any hydrophobic amino acid; and X₃ is a sequence of amino acidresidues of between 0 and 200 residues in length.